Method for producing kakeromycin and derivatives thereof

ABSTRACT

Provided is a production method of kakeromycin and a derivative thereof showing an antifungal activity and cytotoxicity and expected as a new antifungal agent or anticancer agent, by chemical synthesis. A production method of a compound represented by the formula (1):wherein R is an optionally substituted hydrocarbon group or an optionally substituted heterocyclic group; and n is 0 or 1, or a salt thereof, including a step of subjecting a compound represented by the formula (2):wherein R and n are as defined above, or a salt thereof, to an oxidation reaction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional of copending U.S. patentapplication Ser. No. 15/554,002, filed on Aug. 27, 2017, which the U.S.national phase of International Patent Application No.PCT/JP2016/055891, filed on Feb. 26, 2016, which claims the benefit ofJapanese Patent Application No. 2015-039363, filed Feb. 27, 2015, thedisclosures of which are incorporated herein by reference in theirentireties for all purposes.

TECHNICAL FIELD

The present invention relates to a production method of kakeromycin anda derivative thereof.

BACKGROUND ART

In recent years, along with an increase in elderly people, progress ofadvanced medicine, immunodeficiency of late stage cancer patients andthe like, infections with fungi have been increasing. These infectionsprovide serious effects, often causing death. Since there are not manykinds of existing antifungal agents, and their toxicity is high, themother nucleus of a new antifungal agent, which is different from thatof conventional medicaments, has been desired. In addition, since theuse of antifungal agents causes increased emergence of resistantbacteria, the development of a new medicament has been earnestlydesired. While candin-based antifungal agents show low toxicity, sincethe molecular weight thereof is large, reactivity with serum posesproblems. Azole-based antifungal agents have a problem in thatadministration at a high concentration is difficult in view of thetoxicity thereof. Therefore, an effective, low-molecular-weight compoundshowing low reactivity with serum and low toxicity has been stronglydesired.

Conventionally, in search of a pharmaceutical product seed compound frommicrobial metabolites, terrestrial separation sources have been mainlyharvested and subjected to microorganism separation. The microbialmetabolites found to date include penicillin and adriamycin, and anumber of antibiotics and anticancer agents were found and utilized astherapeutic drugs for infection, cancer and the like. However, due tothe continuous search over a long term, microbial metabolites obtainedfrom the land areas are mostly known compounds, and a secondarymetabolite to be a candidate for a novel medicament is extremelydifficult to obtain. Consequently, the development of a novel medicamentby natural substance drug discovery corporations was rapidly reduced. Toovercome the situation, screening using a chemical library (naturalsubstance and synthesized compound) has been conducted on a globalscale. Unexpectedly, however, a promising novel medicament candidatecompound was not obtained from the chemical library. Under suchcircumstances, it is extremely difficult to obtain a new medicamentcandidate compound.

In view of the aforementioned current situation in the search of a novelmedicament candidate compound, the marine microorganism resources havedrawing attention. Marine microorganism resources have been scarcelyutilized, and have a high possibility of affording a novel secondarymetabolite.

Recently, a new compound represented by the following formula:

which was found from a microorganism collected from the seabed sandaround the Kakeroma island of Kagoshima Prefecture, Amami Islands, wasnamed “Kakeromycin”. The “kakeromycin” shows an antifungal activity,particularly, a strong antibacterial activity against pathogens ofcandidiasis, highly possibly shows a new antibacterial action differentfrom those of existing antifungal agents, and further research anddevelopment in the future is expected. In addition, since the“kakeromycin” shows cytotoxicity to HepG2 liver cancer cell and PANC-1pancreas cancer cell, its development as an anticancer agent isexpected.

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method of producingkakeromycin and a derivative thereof by chemical synthesis.

Means of Solving the Problems

The present inventors have conducted intensive studies in an attempt tosolve the above-mentioned problem and found a method of producingkakeromycin and a derivative thereof represented by the followingformula (1) by chemical synthesis, which resulted in the completion ofthe present invention.

Therefore, the present invention provides the following.

-   [1] A method of producing a compound represented by the formula (1):

-   wherein-   R is an optionally substituted hydrocarbon group or an-   optionally substituted heterocyclic group; and-   n is 0 or 1,-   or a salt thereof, which comprises a step of subjecting a compound    represented by the formula (2):

-   wherein R and n are as defined above,-   or a salt thereof to an oxidation reaction;-   [2] the production method of [1], further comprising a step of    producing a compound represented by the formula (2) or a salt    thereof by subjecting a compound represented by the formula (3):

-   wherein R and n are as defined in [1],-   or a salt thereof, to an intramolecular dehydration condensation    reaction;-   [3] the production method of [2], further comprising a step of    producing a compound represented by the formula (3) or a salt    thereof by subjecting a compound represented by the formula (4):

-   wherein R and n are as defined in [1],-   or a salt thereof, to an intramolecular addition reaction;-   [4] the production method of [3], further comprising a step of    producing a compound represented by the formula (4) or a salt    thereof by subjecting an acyl protecting group of an amino group of    a compound represented by the formula (5):

-   wherein R⁴ is an optionally substituted hydrocarbon group or an    optionally substituted hydrocarbon-oxy group, and R and n are as    defined in [1],-   or a salt thereof, to a deprotection reaction;-   [5] the production method of [4], further comprising a step of    producing a compound represented by the formula (5) or a salt    thereof by subjecting a compound represented by the formula (6):

-   wherein R and n are as defined in [1], and R⁴ is as defined in [4],-   or a salt thereof, to a reduction reaction;-   [6] the production method of [5], further comprising a step of    producing a compound represented by the formula (6) or a salt    thereof by subjecting a compound represented by the formula (7):

-   wherein n is as defined in [1], and R⁴ is as defined in [4], or a    salt thereof, and a compound represented by the formula (8):

-   wherein R is as defined in [1],-   or a salt thereof, to a cyclization addition reaction;-   [7] the production method of any one of [1]-[6], further comprising    a step of producing a compound represented by the formula (1-1):

-   wherein R³ is an optionally substituted hydrocarbon group or an    optionally substituted acyl group, and R and n are as defined in    [1],-   or a salt thereof, by subjecting a hydroxyl group of a compound    represented by the formula (1) or a salt thereof to a protection    reaction;-   [8] the production method of any one of [1]-[7], wherein R is a    group represented by the formula (A):

-   wherein R¹ and R² are the same or different and each is an    optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group;-   [9] the production method of any one of [1]-[8], wherein n is 1;-   [10] a compound represented by the formula (1):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group; and-   n is 0 or 1,-   or a salt thereof;-   [11] the compound of [10], wherein R is a group represented by the    formula (A):

-   wherein R¹ and R² are the same or different and each is an    optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group,-   or a salt thereof;-   [12] a compound represented by the formula (2):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group; and-   n is 0 or 1,-   or a salt thereof;-   [13] a compound represented by the formula (3):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group; and-   n is 0 or 1,-   or a salt thereof;-   [14] a compound represented by the formula (4):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group; and-   n is 0 or 1,-   or a salt thereof;-   [15] a compound represented by the formula (5):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group;-   R⁴ is an optionally substituted hydrocarbon group or an optionally    substituted hydrocarbon-oxy group; and-   n is 0 or 1,-   or a salt thereof;-   [16] a compound represented by the formula (6):

-   wherein-   R is an optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group;-   R⁴ is an optionally substituted hydrocarbon group or an optionally    substituted hydrocarbon-oxy group; and-   n is 0 or 1,-   or a salt thereof.

Effect of the Invention

According to the production method of the present invention, kakeromycinand a derivative thereof which show an antifungal activity andcytotoxicity, and are expected as new antifungal agents and anticanceragents can be produced by chemical synthesis.

DESCRIPTION OF EMBODIMENTS

The definition of each group used in the structural formulas in thepresent specification is described in detail below.

R, R¹ and R² are each an optionally substituted hydrocarbon group or anoptionally substituted heterocyclic group.

R³ is an optionally substituted hydrocarbon group or an optionallysubstituted acyl group.

R⁴ is an optionally substituted hydrocarbon group or an optionallysubstituted hydrocarbon-oxy group.

Examples of the “hydrocarbon group” of the “optionally substitutedhydrocarbon group” and the “hydrocarbon-” (hydrocarbon moiety) of the“optionally substituted hydrocarbon-oxy group” include C₁₋₂₀ alkylgroup, C₂₋₂₀ alkenyl group, C₂₋₂₀ alkynyl group, C₃₋₂₀ cycloalkyl group,C₃₋₂₀ cycloalkenyl group, C₆₋₂₀ aryl group, and C₇₋₂₀ aralkyl group.

Examples of the “C₁₋₂₀ alkyl group” include methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl,neopentyl, 1-ethylpropyl, hexyl, isohexyl, 1,1-dimethylbutyl,2,2-dimethylbutyl, 3,3-dimethylbutyl, and 2-ethylbutyl.

Examples of the “C₂₋₂₀ alkenyl group” include ethenyl, 1-propenyl,2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, 3-butenyl,3-methyl-2-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,4-methyl-3-pentenyl, 1-hexenyl, 3-hexenyl, and 5-hexenyl.

Examples of the “C₂₋₂₀ alkynyl group” include ethynyl, 1-propynyl,2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl,3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl,5-hexynyl, and 4-methyl-2-pentynyl.

Examples of the “C₃₋₂₀ cycloalkyl group” include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, bicyclo[2.2.1]heptyl, bicyclo [2.2.2]octyl, bicyclo [3.2.1]octyl, andadamantyl.

Examples of the “C₃₋₂₀ cycloalkenyl group” include cyclopropenyl,cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andcyclooctenyl.

Examples of the “C₆₋₂₀ aryl group” include phenyl, 1-naphthyl,2-naphthyl, 1-anthryl, 2-anthryl, and 9-anthryl.

Examples of the “C₇₋₂₀ aralkyl group” include benzyl, phenethyl,naphthylmethyl, and phenylpropyl.

Examples of the “heterocyclic group” of the “optionally substitutedheterocyclic group” include (i) aromatic heterocyclic group, (ii)nonaromatic heterocyclic group and (iii) 7- to 10-membered crosslinkedheterocyclic group, each containing, as a ring-constituting atom besidescarbon atom, 1 to 4 hetero atoms selected from nitrogen atom, sulfuratom and oxygen atom.

Examples of the “aromatic heterocyclic group” include a 5- to14-membered (preferably 5- to 10-membered) aromatic heterocyclic groupcontaining, as a ring-constituting atom besides carbon atom, 1 to 4hetero atoms selected from nitrogen atom, sulfur atom and oxygen atom.

Preferable examples of the “aromatic heterocyclic group” include 5- or6-membered monocyclic aromatic heterocyclic groups such as thienyl,furyl, pyrrolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl,oxazolyl, isoxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl,1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,4-thiadiazolyl,1,3,4-thiadiazolyl, triazolyl, tetrazolyl, triazinyl and the like; and8- to 14-membered condensed polycyclic (preferably di- or tricyclic)aromatic heterocyclic groups such as benzothiophenyl, benzofuranyl,benzimidazolyl, benzoxazolyl, benzoisooxazolyl, benzothiazolyl,benzoisothiazolyl, benzotriazolyl, imidazopyridinyl, thienopyridinyl,furopyridinyl, pyrrolopyridinyl, pyrazolopyridinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyrazinyl, imidazopyrimidinyl,thienopyrimidinyl, furopyrimidinyl, pyrrolopyrimidinyl,pyrazolopyrimidinyl, oxazolopyrimidinyl, thiazolopyrimidinyl,pyrazolotriazinyl, naphtho [2,3-b]thienyl, phenoxathiinyl, indolyl,isoindolyl, 1H-indazolyl, purinyl, isoquinolyl, quinolyl, phthalazinyl,naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, carbazolyl,β-carbolinyl, phenanthridinyl, acrydinyl, phenazinyl, phenothiazinyl,phenoxazinyl and the like.

Examples of the “nonaromatic heterocyclic group” include a 3- to14-membered (preferably 4- to 10-membered) nonaromatic heterocyclicgroup containing, as a ring-constituting atom besides carbon atom, 1 to4 hetero atoms selected from nitrogen atom, sulfur atom and oxygen atom.

Preferable examples of the “nonaromatic heterocyclic group” include 3-to 8-membered monocyclic nonaromatic heterocyclic groups such asaziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl,tetrahydrothienyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl,imidazolinyl, imidazolidinyl, oxazolinyl, oxazolidinyl, pyrazolinyl,pyrazolidinyl, thiazolinyl, thiazolidinyl, tetrahydroisothiazolyl,tetrahydrooxazolyl, tetrahydroisooxazolyl, piperidinyl, piperazinyl,tetrahydropyridinyl, dihydropyridinyl, dihydrothiopyranyl,tetrahydropyrimidinyl, tetrahydropyridazinyl, dihydropyranyl,tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, thiomorpholinyl,azepanyl, diazepanyl, azepinyl, oxepanyl, azocanyl, diazocanyl and thelike; and 9- to 14-membered condensed polycyclic (preferably di- ortricyclic) nonaromatic heterocyclic groups such as dihydrobenzofuranyl,dihydrobenzoimidazolyl, dihydrobenzooxazolyl, dihydrobenzothiazolyl,dihydrobenzoisothiazolyl, dihydronaphtho [2,3-b]thienyl,tetrahydroisoquinolyl, tetrahydroquinolyl, 4H-quinolizinyl, indolinyl,isoindolinyl, tetrahydrothieno [2,3-c]pyridinyl,tetrahydrobenzoazepinyl, tetrahydroquinoxalinyl,tetrahydrophenanthridinyl, hexahydrophenothiazinyl,hexahydrophenoxazinyl, tetrahydrophthalazinyl, tetrahydronaphthyridinyl,tetrahydroquinazolinyl, tetrahydrocinnolinyl, tetrahydrocarbazolyl,tetrahydro-β-carbolinyl, tetrahydroacrydinyl, tetrahydrophenazinyl,tetrahydrothioxanthenyl, octahydroisoquinolyl and the like.

Preferable examples of the “7- to 10-membered crosslinked heterocyclicgroup” include quinuclidinyl, and 7-azabicyclo [2.2.1]heptanyl.

Examples of the “acyl group” of the “optionally substituted acyl group”include formyl group, carboxy group, C₁₋₆ alkyl-carbonyl group, C₂₋₆alkenyl-carbonyl group, C₃₋₁₀ cycloalkyl-carbonyl group, C₃₋₁₀cycloalkenyl-carbonyl group, C₆₋₁₄ aryl-carbonyl group, C₇₋₁₆aralkyl-carbonyl group, aromatic heterocyclyl-carbonyl group,non-aromatic heterocyclyl-carbonyl group, C₁₋₆ alkoxy-carbonyl group,C₆₋₁₄ aryloxy-carbonyl group, C₇₋₁₆ aralkyloxy-carbonyl group, andcarbamoyl group.

Examples of the “C₁₋₆ alkyl-carbonyl group” include acetyl, propanoyl,butanoyl, 2-methylpropanoyl, pentanoyl, 3-methylbutanoyl,2-methylbutanoyl, 2,2-dimethylpropanoyl, hexanoyl, and heptanoyl.

Examples of the “C₂₋₆ alkenyl-carbonyl group” include ethenylcarbonyl,1-propenylcarbonyl, 2-propenylcarbonyl, 2-methyl-1-propenylcarbonyl,1-butenylcarbonyl, 2-butenylcarbonyl, 3-butenylcarbonyl,3-methyl-2-butenylcarbonyl, 1-pentenylcarbonyl, 2-pentenylcarbonyl,3-pentenylcarbonyl, 4-pentenylcarbonyl, 4-methyl-3-pentenylcarbonyl,1-hexenylcarbonyl, 3-hexenylcarbonyl, and 5-hexenylcarbonyl.

Examples of the “C₃₋₂₀ cycloalkyl-carbonyl group” includecyclopropylcarbonyl, cyclobutylcarbonyl, cyclopentylcarbonyl,cyclohexylcarbonyl, cycloheptylcarbonyl, cyclooctylcarbonyl, bicyclo[2.2.1]heptylcarbonyl, bicyclo [2.2.2]octylcarbonyl, bicyclo[3.2.1]octylcarbonyl, and adamantylcarbonyl.

Examples of the “C₃₋₂₀ cycloalkenyl-carbonyl group” includecyclopropenylcarbonyl, cyclobutenylcarbonyl, cyclopentenylcarbonyl,cyclohexenylcarbonyl, cycloheptenylcarbonyl, and cyclooctenylcarbonyl.

Examples of the “C₆₋₁₄ aryl-carbonyl group” include benzoyl,1-naphthoyl, and 2-naphthoyl.

Examples of the “C₇₋₁₆ aralkyl-carbonyl group” include phenylacetyl, andphenylpropionyl.

Examples of the “aromatic heterocyclyl-carbonyl group” includenicotinoyl, isonicotinoyl, thenoyl, and furoyl.

Examples of the “non-aromatic heterocyclyl-carbonyl group” includemorpholinylcarbonyl, piperidinylcarbonyl, and pyrrolidinylcarbonyl.

Examples of the “C₁₋₆ alkoxy-carbonyl group” include methoxycarbonyl,ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl,isobutoxycarbonyl, sec-butoxycarbonyl, tert-butoxycarbonyl,pentyloxycarbonyl, and hexyloxycarbonyl.

Examples of the “C₆₋₁₄ aryloxy-carbonyl group” includephenyloxycarbonyl, 1-naphthyloxycarbonyl, and 2-naphthyloxycarbonyl.

Examples of the “C₇₋₁₆ aralkyloxy-carbonyl group” includebenzyloxycarbonyl, and phenethyloxycarbonyl.

Examples of the “substituent” of the “optionally substituted hydrocarbongroup”, “optionally substituted hydrocarbon-oxy group”, “optionallysubstituted heterocyclic group” and “optionally substituted acyl group”include the following:

-   (1) halogen atom,-   (2) nitro group,-   (3) cyano group,-   (4) oxo group,-   (5) hydroxy group,-   (6) optionally substituted C₁₋₆ alkoxy group,-   (7) optionally substituted C₆₋₁₄ aryloxy group,-   (8) optionally substituted C₇₋₁₆ aralkyloxy group,-   (9) optionally substituted aromatic heterocyclyl-oxy group,-   (10) optionally substituted non-aromatic heterocyclyl-oxy group,-   (11) optionally substituted C₁₋₆ alkyl-carbonyloxy group,-   (12) optionally substituted C₆₋₁₄ aryl-carbonyloxy group,-   (13) optionally substituted C₁₋₆ alkoxy-carbonyloxy group,-   (14) optionally substituted mono- or di-C₁₋₆ alkyl-carbamoyloxy    group,-   (15) optionally substituted C₆₋₁₄ aryl-carbamoyloxy group,-   (16) optionally substituted 5- to 14-membered aromatic    heterocyclyl-carbonyloxy group,-   (17) optionally substituted 3- to 14-membered non-aromatic    heterocyclyl-carbonyloxy group,-   (18) optionally substituted C₁₋₆ alkylsulfonyloxy group,-   (19) optionally substituted C₆₋₁₄ arylsulfonyloxy group,-   (20) optionally substituted C₁₋₆ alkylthio group,-   (21) optionally substituted 5- to 14-membered aromatic heterocyclic    group,-   (22) optionally substituted 3- to 14-membered nonaromatic    heterocyclic group,-   (23) formyl group,-   (24) carboxy group,-   (25) optionally substituted C₁₋₆ alkyl-carbonyl group,-   (26) optionally substituted C₆₋₁₄ aryl-carbonyl group,-   (27) optionally substituted 5- to 14-membered aromatic    heterocyclyl-carbonyl group,-   (28) optionally substituted 3- to 14-membered non-aromatic    heterocyclyl-carbonyl group,-   (29) optionally substituted C₁₋₆ alkoxy-carbonyl group,-   (30) optionally substituted C₆₋₁₄ aryloxy-carbonyl group,-   (31) optionally substituted C₇₋₁₆ aralkyloxy-carbonyl group,-   (32) carbamoyl group,-   (33) thiocarbamoyl group,-   (34) optionally substituted mono- or di-C₁₋₆ alkyl-carbamoyl group,-   (35) optionally substituted C₆₋₁₄ aryl-carbamoyl group,-   (36) optionally substituted 5- to 14-membered aromatic    heterocyclyl-carbamoyl group,-   (37) optionally substituted 3- to 14-membered non-aromatic    heterocyclyl-carbamoyl group,-   (38) optionally substituted C₁₋₆ alkylsulfonyl group,-   (39) optionally substituted C₆₋₁₄ arylsulfonyl group,-   (40) optionally substituted 5- to 14-membered aromatic    heterocyclyl-sulfonyl group,-   (41) optionally substituted C₁₋₆ alkylsulfinyl group,-   (42) optionally substituted C₆₋₁₄ arylsulfinyl group,-   (43) optionally substituted 5- to 14-membered aromatic    heterocyclyl-sulfinyl group,-   (44) amino group,-   (45) optionally substituted mono- or di-C₁₋₆ alkylamino group,-   (46) optionally substituted mono- or di-C₆₋₁₄ arylamino group,-   (47) optionally substituted 5- to 14-membered aromatic    heterocyclyl-amino group,-   (48) optionally substituted C₇₋₁₆ aralkylamino group,-   (49) formylamino group,-   (50) optionally substituted C₁₋₆ alkyl-carbonylamino group,-   (51) optionally substituted (C₁₋₆ alkyl) (C₁₋₆ alkyl-carbonyl)amino    group,-   (52) optionally substituted C₆₋₁₄ aryl-carbonylamino group,-   (53) optionally substituted C₁₋₆ alkoxy-carbonylamino group,-   (54) optionally substituted C₇₋₁₆ aralkyloxy-carbonylamino group,-   (55) optionally substituted C₁₋₆ alkylsulfonylamino group,-   (56) optionally substituted C₆₋₁₄ arylsulfonylamino group,-   (57) optionally substituted C₁₋₆ alkyl group,-   (58) optionally substituted C₂₋₆ alkenyl group,-   (59) optionally substituted C₂₋₆ alkynyl group,-   (60) optionally substituted C₃₋₁₀ cycloalkyl group,-   (61) optionally substituted C₃₋₁₀ cycloalkenyl group, and-   (62) optionally substituted C₆₋₁₄ aryl group.

The number of the above-mentioned “substituent” of the “optionallysubstituted hydrocarbon group”, “optionally substituted hydrocarbon-oxygroup”, “optionally substituted heterocyclic group” and “optionallysubstituted acyl group” is, for example, 1 to 5, preferably 1 to 3. Whenthe number of the substituents is two or more, the respectivesubstituents may be the same or different.

R is preferably an optionally substituted C₂₋₂₀ alkenyl group (e.g.,ethenyl), more preferably, a group represented by the formula (A):

-   wherein R¹ and R² are the same or different and each is an    optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group, more preferably, a group represented    by the formula (A) wherein R¹ and R² are the same or different and    each is an optionally substituted C₁₋₂₀ alkyl group (e.g., methyl)    or an optionally substituted C₇₋₂₀ aralkyl group (e.g., benzyl),    particularly preferably, a group represented by the formula (A)    wherein R¹ and R² are the same or different and each is a C₁₋₂₀    alkyl group (e.g., methyl) or a C₇₋₂₀ aralkyl group (e.g., benzyl).

In another embodiment of the present invention, R is preferably anoptionally substituted C₂₋₂₀ alkenyl group (e.g., ethenyl), anoptionally substituted C₁₋₂₀ alkyl group (e.g., heptyl), an optionallysubstituted C₆₋₂₀ aryl group (e.g., phenyl, naphthyl) or an optionallysubstituted C₇₋₂₀ aralkyl group (e.g., phenylethyl), more preferably, agroup represented by the formula (A):

-   wherein R¹ and R² are the same or different and each is an    optionally substituted hydrocarbon group or an optionally    substituted heterocyclic group, an optionally substituted C₁₋₂₀    alkyl group (e.g., heptyl), an optionally substituted C₆₋₂₀ aryl    group (e.g., phenyl, naphthyl) or an optionally substituted C₇₋₂₀    aralkyl group (e.g., phenylethyl), more preferably, a group    represented by the formula (A) wherein R¹ and R² are the same or    different and each is an optionally substituted C₁₋₂₀ alkyl group    (e.g., methyl), an optionally substituted C₆₋₂₀ aryl group (e.g.,    phenyl) or an optionally substituted C₇₋₂₀ aralkyl group (e.g.,    benzyl, phenylethyl), an optionally substituted C₁₋₂₀ alkyl group    (e.g., heptyl), an optionally substituted C₆₋₂₀ aryl group (e.g.,    phenyl, naphthyl) or an optionally substituted C₇₋₂₀ aralkyl group    (e.g., phenylethyl), particularly preferably, a group represented by    the formula (A) wherein R¹ and R² are the same or different and each    is a C₁₋₂₀ alkyl group (e.g., methyl), a C₆₋₂₀ aryl group (e.g.,    phenyl) optionally substituted by a halogen atom (e.g., chlorine    atom) or C₇₋₂₀ aralkyl group (e.g., benzyl, phenylethyl), a C₁₋₂₀    alkyl group (e.g., heptyl), a C₆₋₂₀ aryl group (e.g., phenyl,    naphthyl) or a C₇₋₂₀ aralkyl group (e.g., phenylethyl).

R³ is preferably an optionally substituted C₁₋₂₀ alkyl group (e.g.,methyl) or an optionally substituted C₁₋₆ alkyl-carbonyl group (e.g.,acetyl), more preferably, a C₁₋₂₀ alkyl group (e.g., methyl) or a C₁₋₆alkyl-carbonyl group (e.g., acetyl), further preferably, methyl oracetyl.

R⁴ is preferably an optionally substituted C₁₋₂₀ alkyl group (e.g.,methyl), an optionally substituted C₆₋₂₀ aryl group (e.g., phenyl), anoptionally substituted C₁₋₂₀ alkyl-oxy group (e.g., tert-butyloxy) or anoptionally substituted C₇₋₂₀ aralkyl-oxy group (e.g., benzyloxy), morepreferably a C₁₋₂₀ alkyl group (e.g., methyl), a C₆₋₂₀ aryl group (e.g.,phenyl), a C₁₋₂₀ alkyl-oxy group (e.g., tert-butyloxy) or a C₇₋₂₀aralkyl-oxy group (e.g., benzyloxy), further preferably, methyl, phenyl,tert-butyloxy or benzyloxy, particularly preferably tert-butyloxy.

n is 0 or 1. n is preferably 1.

The production method of the present invention is explained below.

The whole scheme of the production method of the present invention isshown below.

-   wherein each symbol is as defined above.    (Step 1: Production of Oxime 8)

Oxime (8) can be synthesized by a dehydration condensation reaction ofaldehyde (9) and hydroxylamine prepared from hydroxylamine hydrochlorideand a base. As the base, sodium hydrogen carbonate, potassium hydrogencarbonate, sodium carbonate, potassium carbonate, tertiary amines suchas triethylamine and the like, and the like can be used at generally 1-5molar equivalents, preferably 1-1.5 molar equivalents, relative tohydroxylamine hydrochloride, and sodium hydrogen carbonate isparticularly preferable. Hydroxylamine hydrochloride can be used atgenerally 1-5 molar equivalents, preferably 1-1.5 molar equivalents,relative to aldehyde (9). The reaction temperature is generally 0-50°C., preferably 20-30° C. While the reaction time varies depending on thekind of the reagents, reaction temperature and the like, it is generally1-48 hr, preferably 2-10 hr. As the reaction solvent, THF, water,acetonitrile, ethyl acetate, dichloromethane, or a mixed solvent thereofand the like can be used, and a mixed solvent of THF-water isparticularly preferable.

Aldehyde (9) may be a commercially available product, and can also beproduced according to a method known per se or a method analogousthereto.

(Step 2: Production of Dihydroisoxazole 6A, 6B)

Dihydroisoxazole (6A, 6B) can be synthesized by (3+2) cyclizationaddition reaction of nitrile oxide prepared from oxime (8) and aqueoussodium hypochlorite solution, or oxime (8) and chloramine-T, andN-acylaminobutene (7A) or N-acylaminopropene (7B). The aqueous sodiumhypochlorite solution, or chloramine-T, can be used at generally 1-5molar equivalents, preferably 1-2 molar equivalents, relative to oxime8. Oxime (8) can be used at generally 0.5-3 molar equivalents,preferably 0.8-1.2 molar equivalents, relative to N-acylaminobutene (7A)or N-acylaminopropene (7B). The reaction temperature is generally 0-80°C., preferably 20-30° C. While the reaction time varies depending on thekind of the reagents, reaction temperature and the like, it is generally1-48 hr, preferably 2-10 hr. As the reaction solvent, THF, ethylacetate, dichloromethane, ethanol, methanol, acetonitrile or a mixedsolvent thereof and the like can be used.

N-acylaminobutene (7A) or N-acylaminopropene (7B) may be a commerciallyavailable product, and can also be produced according to a method knownper se or a method analogous thereto.

(Step 3: Production of N-acylaminohydroxyketone 5A, 5B)

N-acylaminohydroxyketone (5A, 5B) can be synthesized by reducing N—Obond of dihydroisoxazole (6A, 6B). As the reducing agent, molybdenumhexacarbonyl, cobalt octacarbonyl, iron, zinc, magnesium and the likecan be used at generally 1-5 molar equivalents, preferably 1-2 molarequivalents, relative to dihydroisoxazole (6A, 6B), though subject tochange depending on the kind of the reagents, reaction temperature andthe like, and molybdenum hexacarbonyl is particularly preferable. Thereaction temperature is generally 0-100° C., preferably 70-90° C. Whilethe reaction time varies depending on the kind of the reagents, reactiontemperature and the like, it is generally 1-24 hr, preferably 1-3 hr. Asthe reaction solvent, acetonitrile, propionitrile, water, THF, ethylacetate, dichloromethane, dichloroethane, or a mixed solvent thereof andthe like can be used, and a mixed solvent of acetonitrile-water isparticularly preferable.

(Step 4: Production of Aminohydroxyketone 4A, 4B)

Aminohydroxyketone (4A, 4B) can be synthesized by removing an acylprotecting group of the amino group of N-acylaminohydroxyketone (5A,5B). As the deprotecting agent, trifluoroacetic acid, hydrochloric acid,sodium hydroxide, potassium hydroxide and the like can be used atgenerally 1-50 molar equivalents, preferably 1-10 molar equivalents,relative to N-acylaminohydroxyketone (5A, 5B), and trifluoroacetic acidis particularly preferable. The reaction temperature is generally 0-50°C., preferably 20-30° C. While the reaction time varies depending on thekind of the reagents, reaction temperature and the like, it is generally1-24 hr, preferably 1-3 hr. As the reaction solvent, THF, ethyl acetate,dichloromethane, dichloroethane, or a mixed solvent thereof and the likecan be used, and particularly, dichloromethane and dichloroethane arepreferable.

(Step 5: Production of Cyclic Hemiaminal 3A, 3B)

Cyclic hemiaminal (3A, 3B) can be synthesized by an intramolecularaddition reaction of aminohydroxyketone (4A, 4B). While anintramolecular addition reaction sometimes proceeds without particularlyusing a reaction agent, when an acid catalyst is necessary,trifluoroacetic acid, acetic acid, p-toluenesulfonic acid,methanesulfonic acid and the like, can be used at generally 0.01-5 molarequivalents, preferably 0.01-1 equimolar amount, relative toaminohydroxyketone (4A, 4B). The reaction temperature is generally0-100° C., preferably 30-50° C. While the reaction time varies dependingon the kind of the reagents, reaction temperature and the like, it isgenerally 1-24 hr, preferably 1-6 hr. As the reaction solvent, THF,ethyl acetate, dichloromethane, dichloroethane, toluene, or a mixedsolvent thereof and the like can be used, and THF or dichloroethane isparticularly preferable.

(Step 6: Production of Cyclic Imine 2A, 2B)

Cyclic imine (2A, 2B) can be synthesized by an intramoleculardehydration condensation reaction of cyclic hemiaminal (3A, 3B). As thedehydrating agent, trifluoroacetic acid, acetic acid, molecular sieve,anhydrous sodium sulfate and the like can be used at generally 0.01-100molar equivalents, preferably 0.01-10 molar equivalents, relative tocyclic hemiaminal (3A, 3B), though subject to change depending on thekind of the reagents, reaction temperature and the like. The reactiontemperature is generally 0-100° C., preferably 20-60° C. While thereaction time varies depending on the kind of the reagents, reactiontemperature and the like, it is generally 1-24 hr, preferably 3-12 hr.As the reaction solvent, THF, ethyl acetate, dichloromethane,dichloroethane, toluene, or a mixed solvent thereof and the like can beused, and THF is particularly preferable.

(Step 7: Production of Bicyclic Oxaziridine 1A, 1B)

Bicyclic oxaziridine (1A, 1B) can be synthesized by an oxidationreaction of cyclic imine (2A, 2B). As the oxidant, m-chloroperbenzoicacid, peracetic acid and the like can be used at generally 1-5 molarequivalents, preferably 1-2 molar equivalents, relative to cyclic imine(2A, 2B), and m-chloroperbenzoic acid is particularly preferable. Thereaction temperature is generally 0-50° C., preferably 10-30° C. Whilethe reaction time varies depending on the kind of the reagents, reactiontemperature and the like, it is generally 0.5-12 hr, preferably 1-2 hr.As the reaction solvent, THF, ethyl acetate, dichloromethane,dichloroethane, toluene, ethanol, methanol, acetonitrile, or a mixedsolvent thereof and the like can be used, and THF and dichloromethaneare particularly preferable.

(Step 8: Production of Bicyclic Oxaziridine Derivative 1-1A, 1-1B)

The bicyclic oxaziridine derivative (1-1A, 1-1B) can be synthesized byprotecting the hydroxyl group of bicyclic oxaziridine (1A, 1B).

When R³ is an optionally substituted hydrocarbon group, the protectionreaction can be performed using the corresponding halide as a protectorand a base each at generally 1-10 molar equivalents, preferably 1-3molar equivalents, relative to the bicyclic oxaziridine derivative(1-1A, 1-1B). As the halide, methyl iodide is particularly preferable.As the base, sodium carbonate, potassium carbonate, sodium hydride,n-butyllithium and the like can be used, and sodium hydride isparticularly preferable. The reaction temperature is generally 0-50° C.,preferably 20-30° C. While the reaction time varies depending on thekind of the reagents, reaction temperature and the like, it is generally0.5-24 hr, preferably 1-12 hr. As the reaction solvent, THF, dimethylsulfoxide, dimethylformamide, acetonitrile, dichloromethane,dichloroethane, or a mixed solvent thereof and the like can be used, anddimethyl sulfoxide or acetonitrile is particularly preferable.

When R³ is an optionally substituted acyl group, the protection reactioncan be performed using the corresponding halogenated acyl compound oracid anhydride as a protector at generally 1-10 molar equivalents,preferably 1-3 molar equivalents, relative to the bicyclic oxaziridinederivative (1-1A, 1-1B) in the presence of an amine base. As thehalogenated acyl compound or acid anhydride, acetic anhydride isparticularly preferable. As the amine base, a tertiary amine such astriethylamine, diisopropylethylamine and the like, or a pyridinederivative such as pyridine, dimethylaminopyridine and the like can beused at generally 1-10 molar equivalents, preferably 1-3 molarequivalents, relative to the bicyclic oxaziridine derivative (1-1A,1-1B), and triethylamine or dimethylaminopyridine is particularlypreferable. The reaction temperature is generally 0-50° C., preferably20-30° C. While the reaction time varies depending on the kind of thereagents, reaction temperature and the like, it is generally 0.5-24 hr,preferably 1-12 hr. As the reaction solvent, THF, ethyl acetate,acetonitrile, dichloromethane, dichloroethane, or a mixed solventthereof and the like can be used, and THF or dichloromethane isparticularly preferable.

The kakeromycin derivatives represented by the formulas (1) and (1-1)(excluding kakeromycin) obtained by the production method of the presentinvention, as well as synthetic intermediates thereof represented by theformulas (2), (3), (4), (5) and (6) are novel compounds.

The kakeromycin and a derivative thereof (bicyclic oxaziridine and aderivative thereof) and a synthetic intermediate thereof obtained by theproduction method of the present invention may be salts. Examples ofsuch salt include salts with inorganic acids such as hydrochloric acid,hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid and thelike, and salts with organic acids such as acetic acid, phthalic acid,fumaric acid, oxalic acid, tartaric acid, maleic acid, citric acid,succinic acid, methanesulfonic acid, p-toluenesulfonic acid and thelike. Of these salts, a pharmaceutically acceptable salt is preferable.

The kakeromycin and a derivative thereof (bicyclic oxaziridine and aderivative thereof) obtained by the production method of the presentinvention have a strong antifungal activity against a broad range offungi, and are expected to be new antifungal agents. In addition, thekakeromycin and a derivative thereof show cytotoxicity against cancercells. Therefore, a compound containing the kakeromycin or a derivativethereof as an active ingredient can be used as a medicament, a pesticideand the like.

Examples of the fungi to be the target of the antifungal agent include,but are not limited to, fungi such as the genus Candida (e.g., Candidaalbicans, Candida parapsilosis, Candida tropicalis, Candida krusei,Candida glabrata, Candida quilliermondii, Candida lusitaniae etc.), thegenus Aspergillus (e.g., Aspergillus fumigatus, Aspergillus flavus,Aspergillus niger, Aspergillus terreus etc.), the genus Trichophyton(e.g., Trichophyton rubrum, Trichophyton mentagrophytes, Trichophytontonsurans, Microsporum canis, Microsporum gypseum, Trichophytonverrucosum etc.) and the like. Mycosis is not particularly limited, anddeep skin mycosis, deep mycosis, mycetoma, and fungemia can bementioned.

When the antifungal agent is used as a pesticide, the target crop is notparticularly limited and, for example, plants such as grain (e.g., rice,barley, wheat, rye, oats, corn, kaoliang etc.), beans (soybean, adzukibean, broad bean, pea, peanut etc.), fruit-tree, fruits (apple, citrus,pear, grapes, peach, ume (Japanese plum), cherry, walnut, almond,banana, strawberry etc.), vegetables (cabbage, tomato, spinach,broccoli, lettuce, onion, green onion, bell pepper etc.), rootvegetables (carrot, potato, sweet potato, radish, lotus root, turnipetc.), crops for processing (cotton, hemp, kozo (paper mulberry),mitsumata plant, rape seed, beet, hop, sugarcane, sugar beet, olive,rubber, coffee, tobacco, tea etc.), gourds (pumpkin, cucumber,watermelon, melon etc.), grasses (orchard grass, sorghum, timothy,clover, alfalfa etc.), sods (Korean lawn grass, bentgrass etc.), cropsfor flavor etc. (lavender, rosemary, thyme, parsley, pepper, gingeretc.), flowering plants (chrysanthemum, rose, orchid etc.) and the likecan be mentioned. The antifungal agent can be used for controlling thediseases related to the aforementioned fungi in the crops, by treatingthe target crop and/or seed of the target crop with an effective amountthereof.

The pesticide can be used at the following form, and generally usedtogether with an adjuvant conventionally used in the pharmaceuticalfields. The kakeromycin and a derivative thereof obtained by theproduction method of the present invention are formulated by a knownmethod into, for example, emulsion stock solution, sprayable paste,sprayable or dilutable solution, dilutable emulsion, wettable agent,water soluble powder, powder, granule, flowable pesticide, dry flowablepesticide, smoking agent, fumigant and, for example, capsule made of apolymer substance.

As additive and carrier when the object is a solid agent, plant-derivedpowder such as soy flour, wheat flour and the like, mineral fine powdersuch as diatomaceous earth, apatite, plaster, talc, bentonite, clay andthe like, and organic and inorganic compounds such as sodium benzoate,urea, salt cake and the like can be used.

When a liquid dosage form is desired, vegetable oil, mineral oil,kerosene, aromatic hydrocarbons such as xylene and toluene, amides suchas formamide, and dimethylformamide, sulfoxides such as dimethylsulfoxide, ketones such as methyl isobutyl ketone and acetone,trichloroethylene, water and the like are used as solvents. To affordthese preparations in a uniform and stable form, a surfactant can alsobe added where necessary. The thus-obtained wettable agent, emulsion,aqueous solution, flowable pesticide, and dry flowable pesticide arediluted with water to a given concentration and used as a suspension oremulsion, and powder and granule are used by directly spraying on thesoil or plant.

The content and dose of the active ingredient in a pesticide containingthe kakeromycin or a derivative thereof obtained by the productionmethod of the present invention can be changed in a wide range dependingon the dosage form, the kind of fungi to be the application target,target crop and the like.

On the other hand, when the antifungal agent is used as a medicament, itcan be administered to a treatment target, for example, a mammal (e.g.,human, mouse, rat, hamster, rabbit, cat, dog, bovine, sheep, monkeyetc.) by an oral or parenteral administration route (e.g., intravenousinjection, intramuscular injection, subcutaneous administration, rectaladministration, transdermal administration).

When the antifungal agent is transdermally administered, it can contain,besides the above-mentioned active ingredient, oily base, emulsifier andemulsion stabilizer, solubilizing agents, powder component, polymercomponent, adhesiveness improver, film-forming agent, pH adjuster,antioxidant, antiseptic agent, preservative, shape retention agent,moisturizer, skin protector, algefacient, flavor, colorant, chelatingagent, lubricant, blood circulation promoter, astringent, tissue repairpromoter, adiaphoretic, plant extraction component, animal extractioncomponent, anti-inflammatory agent, antipruritic agent and the like asnecessary. As these additives, those generally used for preparations canbe used.

The antifungal agent can be used by formulating the above-mentionedcomponents other than the active ingredient and the like into externaldrugs such as cream, liquid, lotion, emulsion, tincture, ointment,aqueous gel, oily gel, aerosol, powder, shampoo, soap, enamel agent forapplication to nail and the like, by a method conventionally used in thefield of pharmaceutical preparations.

When the antifungal agent is orally administered, it can be preparedinto a dosage form suitable for oral administration such as capsule,tablet, granule, powder, pill, fine granules, troche and the like. Thesepreparations can be produced using additives generally used for oralpreparations, such as excipient, filler, binder, moistening agent,disintegrant, surfactant, lubricant, dispersing agent, buffering agent,preservative, solubilizing agent, antiseptic agent, flavoring agent,soothing agent, stabilizer and the like by a conventional method.

Examples of the cells to be the target of the anticancer agent include,but are not limited to, cancer cells such as HepG2 cell (liver cancercell), PANC1 cell (pancreas cancer cell) and the like. The cancer is notparticularly limited, and brain tumor, skin cancer, leukemia, esophaguscancer, gastric cancer, colorectal cancer, breast cancer, prostatecancer, rectal cancer, osteosarcoma and the like can be mentioned.

EXAMPLES

The present invention is explained in more detail by referring to thefollowing Examples. These do not limit the present invention, and may bechanged within the scope of the present invention.

¹H and ¹³C NMR spectra were measured by a nuclear magnetic resonanceapparatus (manufactured by Varian, 400 MR and Mercury-300), and all δvalues are shown in ppm. Mass spectrum was measured by HPLC-Chip/QTOFmass spectrometry system (Agilent Technologies), and m/z values areshown.

Example 1

(Step 1)

Aldehyde 9a-01 (72 mg, 0.45 mmol) was dissolved in THF (3 mL),hydroxylamine hydrochloride (47 mg, 0.68 mmol), sodium hydrogencarbonate (57 mg, 0.68 mmol) and water (2 mL) were added at roomtemperature, and the mixture was stirred at room temperature for 12 hr.Sodium sulfate (5 g) was added to remove water in the reaction system,and the mixture was filtered through cotton. The obtained filtrate wasconcentrated by a rotary evaporator. The obtained crude product waspurified by silica gel column chromatography (solvent: hexane and ethylacetate) to give oxime 8a-01 (75 mg, 0.43 mmol) as colorless liquid(yield 95%).

¹H NMR (300 MHz, CDCl₃): δ1.85 (d, 3H, J=7.5 Hz), 3.70 (s, 2H), 6.01 (q,1H, J=7.5 Hz), 7.04-7.32 (m, 5H), 7.75 (s, 1H).

(Step 2)

Oxime 8a-01 (52 mg, 0.30 mmol) and N-Boc-aminobutene 7A-01 (62 mg, 0.36mmol)) were dissolved in THF (5 mL), aqueous sodium hypochloritesolution (5%, 2 mL) was added at 0° C., and the mixture was stirred atroom temperature for 12 hr. Sodium sulfate (5 g) was added to removewater in the reaction system, and the mixture was filtered throughcotton. The obtained filtrate was concentrated by a rotary evaporator.The obtained crude product was purified by silica gel columnchromatography (solvent: hexane and ethyl acetate) to givedihydroisoxazole 6Aa-001 (120 mg, 0.35 mmol) as colorless liquid (yield86%).

¹H NMR (300 MHz, CDCl₃): δ1.41 (s, 9H), 1.72-1.86 (m, 2H), 1.87 (d, 3H,J=7.5 Hz), 2.75 (dd, 1H, J=8.1, 16.2 Hz), 3.18 (dd, 1H, J=10.2, 16.2Hz), 3.15-3.30 (m, 2H), 3.80 (s, 2H), 4.55-4.68 (m, 1H), 4.68-4.98 (br,1H), 5.95 (q, 1H, J=7.5 Hz), 7.05-7.28 (m, 5H).

¹³C NMR (75 MHz, CDCl₃): δ14.4, 28.3, 32.6, 35.2, 37.6, 39.8, 79.1,79.6, 125.8, 128.2, 128.4, 131.5, 131.7, 139.6, 156.0, 158.8.

(Step 3)

Dihydroisoxazole 6Aa-001 (110 mg, 0.32 mmol) and molybdenum hexacarbonyl(170 mg, 0.64 mmol) were dissolved in acetonitrile (3 mL), water (0.5mL) was added, and the mixture was stirred at 85° C. for 2 hr. Ethylacetate (5 mL) was added, and the mixture was stirred at roomtemperature for 24 hr. The obtained mixture was filtered through celiteby using ethyl acetate, and the filtrate was concentrated by a rotaryevaporator. The obtained crude product was purified by silica gel columnchromatography (solvent: hexane and ethyl acetate) to give N-Bocaminohydroxyketone 5Aa-001 (83 mg, 0.24 mmol) as colorless liquid (yield75%).

¹H NMR (300 MHz, CDCl₃): δ1.20-1.62 (m, 2H), 1.43 (s, 9H), 1.96 (d, 3H,J=7.5 Hz), 2.78-2.83 (m, 2H), 3.10-3.41 (m, 2H), 3.68 (s, 2H), 4.16-4.28(m, 1H), 4.75-5.10 (br, 1H), 6.94 (q, 1H, J=7.5 Hz), 7.08-7.24 (m, 5H).

(steps 4 and 5)

N-Boc aminohydroxyketone 5Aa-001 (50 mg, 0.14 mmol) was dissolved indichloroethane (3 mL), trifluoroacetic acid (0.5 mL) was added, and themixture was stirred at room temperature for 1 hr. The reaction mixturewas concentrated by a rotary evaporator to give a mixture (31 mg, 0.13mol) of aminohydroxyketone 4Aa-001 and cyclic hemiaminal 3Aa-001 asyellow liquid (yield 87%).

¹H NMR (300 MHz, CDCl₃): δ1.20-1.64 (m, 2H), 1.95 (d, 3H, J=7.5 Hz),2.76-2.96 (m, 4H), 3.67 (s, 2H), 4.12-4.28 (m, 1H), 6.92 (q, 1H, J=7.5Hz), 7.08-7.24 (m, 5H).

(Step 6)

A mixture (30 mg, 0.12 mmol) of aminohydroxyketone 4Aa-001 and cyclichemiaminal 3Aa-001 was dissolved in THF (3 mL), 4A-molecular sieve (100mg) was added, and the mixture was stirred at room temperature for 12hr. The reaction mixture was filtered through celite by using ethylacetate as a solvent, and concentrated by a rotary evaporator to givecyclic imine 2Aa-001 (24 mg, 0.10 mmol) as yellow liquid (yield 83%).

(Step 7)

Cyclic imine 2Aa-001 (9 mg, 0.039 mmol) was dissolved in THF (3 mL),m-chloroperbenzoic acid (11 mg) was added, and the mixture was stirredat room temperature for 3 hr and concentrated by a rotary evaporator.The obtained crude product was purified by silica gel columnchromatography (solvent: hexane and ethyl acetate) to give bicyclicoxaziridine 1Aa-001 (3 mg, 0.012 mmol) as yellow liquid (yield 31%).

¹H NMR (400 MHz, CDCl₃): δ1.16-1.36 (m, 1H), 1.69 (d, 3H, J=7.6 Hz),1.80-1.98 (m, 1H), 1.99 (dd, 1H, J=6.9, 15.1 Hz), 2.35 (ddd, 1H, J=1.3,6.2, 15.1 Hz), 3.15-3.25 (m, 1H), 3.36-3.60 (m, 3H), 3.75-3.84 (m, 1H),5.91 (q, 1H, J=6.9 Hz), 7.05-7.28 (m, 5H).

MS: m/z 246 ([M+1], C₁₅H₁₉NO₂)

Example 2

(Step 2)

In the same manner as in Example 1, Step 2, and by using octylaldehydeoxime 8A-01 and N-Boc-aminobutene 7A-01 as substrates, dihydroisoxazole6A-01 was obtained as colorless liquid (yield 75%).

¹H NMR (300 MHz, CDCl₃): δ0.82 (t, 3H, J=7.5 Hz), 1.16-1.80 (m, 12H),1.39 (s, 9H), 2.20-2.36 (m, 2H), 2.56 (dd, 1H, J=8.1, 16.2 Hz), 2.98(dd, 1H, J=10.2, 16.2 Hz), 3.10-3.24 (m, 2H), 4.48-4.60 (m, 1H),4.92-5.00 (br, 1H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6A-01 as a substrate, N-Boc aminohydroxyketone 5A-01was obtained as colorless liquid (yield 69%).

¹H NMR (300 MHz, CDCl₃): δ0.83 (t, 3H, J=7.5 Hz), 1.16-1.76 (m, 10H),1.40 (s, 9H), 2.32-2.60 (m, 6H), 3.16-3.44 (m, 2H), 4.00-4.26 (m, 1H),4.96-5.04 (br, 1H).

(Steps 4 and 5)

In the same manner as in Example 1, Seps 4 and 5, and by using N-Bocaminohydroxyketone 5A-01 as a substrate, a mixture of aminohydroxyketone4A-01 and cyclic hemiaminal 3A-01 was obtained as yellow liquid (yield90%).

¹H NMR (300 MHz, CDCl₃): δ0.83 (t, 3H, J=7.5 Hz), 1.16-1.76 (m, 10H),2.32-2.60 (m, 6H), 2.64-3.02 (m, 2H), 3.98-4.16 (m, 1H).

(Steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using as a substrate,a mixture of aminohydroxyketone 4A-01 and cyclic hemiaminal 3A-01,cyclic imine 2A-01 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1A-01 was obtained as yellow liquid (yield23%).

¹H NMR (300 MHz, CDCl₃): δ50.83 (t, 3H, J=7.5 Hz), 1.20-1.74 (m, 10H),1.92-2.20 (m, 2H), 2.32-2.60 (m, 4H), 3.78-4.12 (m, 2H), 4.12-4.22 (m,1H).

MS: m/z 214 ([M+1], C₁₂H₂₃NO₂)

Example 3

(Step 2)

In the same manner as in Example 1, Step 2, and by using3-phenylpropionaldehyde oxime 8A-02 and N-Boc-aminobutene 7A-01 assubstrates, dihydroisoxazole 6A-02 was obtained as colorless liquid(yield 77%).

¹H NMR (300 MHz, CDCl₃): δ51.43 (s, 9H), 1.62-1.80 (m, 2H), 2.40-2.66(m, 2H), 2.82-3.02 (m, 4H), 3.16-3.24 (m, 2H), 4.50-4.62 (m, 1H),4.85-4.98 (br, 1H), 7.14-7.48 (m, 5H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6A-02 as a substrate, N-Boc aminohydroxyketone 5A-02was obtained as colorless liquid (yield 69%).

¹H NMR (300 MHz, CDCl₃): δ1.41 (s, 9H), 1.60-1.80 (m, 2H), 2.46-2.62 (m,2H), 2.64-2.94 (m, 4H), 3.04-3.42 (m, 2H), 4.02-4.18 (m, 1H), 4.98-5.04(br, 1H), 7.14-7.32 (m, 5H).

(Steps 4 and 5)

In the same manner as in Steps 4 and 5 of Example 1, and by using N-Bocaminohydroxyketone 5A-02 as a substrate, a mixture of aminohydroxyketone4A-02 and cyclic hemiaminal 3A-02 was obtained as yellow liquid (yield92%).

¹H NMR (300 MHz, CDCl₃): δ1.42 (s, 9H), 1.60-1.80 (m, 2H), 2.46-2.62 (m,2H), 2.62-3.20 (m, 6H), 4.00-4.18 (m, 1H), 7.12-7.34 (m, 5H).

(Steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4A-02 and cyclic hemiaminal 3A-02 as a substrate,cyclic imine 2A-02 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1A-02 was obtained as yellow liquid (yield23%).

¹H NMR (300 MHz, CDCl₃): δ1.58-1.98 (m, 6H), 2.46-2.54 (m, 2H),3.86-4.20 (m, 2H), 4.14-4.24 (m, 1H), 7.22-7.40 (m, 5H).

MS: m/z 220 ([M+1], C₁₃H₁₇NO₂)

Example 4

(Step 2)

In the same manner as in Example 1, Step 2, and by using benzaldehydeoxime 8A-03 and N-Boc-aminobutene 7A-01 as substrates, dihydroisoxazole6A-03 was obtained as colorless liquid (yield 72%).

¹H NMR (300 MHz, CDCl₃): δ1.18-1.64 (m, 2H), 1.43 (s, 9H), 2.78-2.83 (m,2H), 3.08-3.42 (m, 2H), 4.14-4.28 (m, 1H), 4.75-5.10 (br, 1H), 7.20-7.52(m, 5H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6A-03 as a substrate, N-Boc aminohydroxyketone 5A-03was obtained as colorless liquid (yield 67%).

¹H NMR (300 MHz, CDCl₃): δ1.18-1.64 (m, 2H), 1.44 (s, 9H), 2.76-2.84 (m,2H), 3.08-3.42 (m, 2H), 4.15-4.29 (m, 1H), 4.72-5.12 (br, 1H), 7.20-7.52(m, 5H).

(Steps 4 and 5)

In the same manner as in Example 1, Steps 4 and 5, and by using N-Bocaminohydroxyketone 5A-03 as a substrate, a mixture of aminohydroxyketone4A-03 and cyclic hemiaminal 3A-03 was obtained as yellow liquid (yield90%).

¹H NMR (300 MHz, CDCl₃): δ1.16-1.66 (m, 2H), 2.74-2.84 (m, 2H),2.62-2.98 (m, 2H), 4.14-4.28 (m, 1H), 7.20-7.52 (m, 5H).

(Steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4A-03 and cyclic hemiaminal 3A-03 as a substrate,cyclic imine 2A-03 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1A-03 was obtained as yellow liquid (yield25%).

¹H NMR (300 MHz, CDCl₃): δ1.40-1.52 (m, 1H), 1.82-1.98 (m, 1H),2.30-2.48 (m, 1H), 2.78-2.86 (m, 1H), 3.48-3.84 (m, 2H), 4.14-4.22 (m,1H), 7.25-7.50 (m, 5H).

Example 5

(Step 2)

In the same manner as in Example 1, Step 2, and by using2-naphthylaldehyde oxime 8A-04 and N-Boc-aminobutene 7A-01 assubstrates, dihydroisoxazole 6A-04 was obtained as colorless liquid(yield 73%).

¹H NMR (300 MHz, CDCl₃): δ1.45 (s, 9H), 1.84-2.00 (m, 2H), 3.02-3.20 (m,1H), 3.28-3.40 (m, 2H), 3.50-3.62 (m, 1H), 4.78-4.88 (m, 1H), 4.90-5.18(br, 1H), 7.42-8.00 (m, 7H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6A-04 as a substrate, N-Boc aminohydroxyketone 5A-04was obtained as colorless liquid (yield 62%).

¹H NMR (300 MHz, CDCl₃): δ1.43 (s, 9H), 1.62-1.84 (m, 2H), 3.18-3.52 (m,4H), 4.30-4.42 (m, 1H), 5.02-5.18 (br, 1H), 7.42-8.02 (m, 6H), 8.40 (s,1H).

(Steps 4 and 5)

In the same manner as in Example 1, Steps 4 and 5, and by using N-Bocaminohydroxyketone 5A-04 as a substrate, a mixture of aminohydroxyketone4A-04 and cyclic hemiaminal 3A-04 was obtained as yellow liquid (yield91%).

¹H NMR (300 MHz, CDCl₃): δ1.60-1.86 (m, 2H), 2.76-3.50 (m, 4H),4.28-4.40 (m, 1H), 7.40-8.02 (m, 6H), 8.42 (s, 1H).

(Steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4A-04 and cyclic hemiaminal 3A-04 as a substrate,cyclic imine 2A-04 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1A-04 was obtained as yellow liquid (yield24%).

¹H NMR (300 MHz, CDCl₃): δ1.42-1.82 (m, 2H), 1.96-2.26 (m, 2H),3.46-3.86 (m, 2H), 4.13-4.21 (m, 1H), 7.10-8.20 (m, 7H).

MS: m/z 242 ([M+1], C₁₅H₁₅NO₂)

Example 6

(Step 1)

In the same manner as in Example 1, Step 1, and by using aldehyde 9a-02and hydroxylamine hydrochloride as substrates, oxime 8a-02 was obtainedas a colorless solid (yield 90%).

(Step 2)

In the same manner as in Example 1, Step 2, and by using oxime 8a-02 andN-Boc-aminobutene 7A-01 as substrates, dihydroisoxazole 6Aa-02 wasobtained as colorless liquid (yield 71%).

¹H NMR (300 MHz, CDCl₃): δ1.47 (s, 9H), 2.40-2.84 (m, 7H), 3.12-3.40 (m,3H), 3.80 (s, 2H), 4.60-4.74 (m, 1H), 4.76-5.00 (br, 1H), 5.92 (t, 1H,J=6.6 Hz), 7.16-7.40 (m, 10H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6Aa-02 as a substrate, N-Boc aminohydroxyketone 5Aa-002was obtained as colorless liquid (yield 62%).

¹H NMR (300 MHz, CDCl₃): δ1.43 (s, 9H), 2.56-2.82 (m, 7H), 3.02-3.44 (m,3H), 3.82 (s, 2H), 4.02-4.16 (m, 1H), 4.92-5.02 (br, 1H), 6.81 (t, 1H,J=6.6 Hz), 7.10-7.38 (m, 10H).

(Steps 4 and 5)

In the same manner as in Example 1, Steps 4 and 5, and by using N-Bocaminohydroxyketone 5Aa-002 as a substrate, a mixture ofaminohydroxyketone 4Aa-002 and cyclic hemiaminal 3Aa-002 was obtained asyellow liquid (yield 92%).

¹H NMR (300 MHz, CDCl₃): δ2.56-2.82 (m, 7H), 2.94-3.40 (m, 3H), 3.81 (s,2H), 43.98-4.10 (m, 1H), 6.78 (t, 1H, J=6.6 Hz), 7.10-7.40 (m, 10H).

(Steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4Aa-002 and cyclic hemiaminal 3Aa-002 as a substrate,cyclic imine 2Aa-002 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1Aa-002 was obtained as yellow liquid(yield 22%). 5 MS: In/Z 336 ([M+1], C₂₂H₂₅NO₂)

Example 7

(Step 1)

In the same manner as in Example 1, Step 1, and by using aldehyde 9a-03and hydroxylamine hydrochloride as substrates, oxime 8a-03 was obtainedas a colorless solid (yield 85%).

(Step 2)

In the same manner as in Example 1, Step 2, and by using oxime 8a-03 andN-Boc-aminobutene 7A-01 as substrates, dihydroisoxazole 6Aa-03 wasobtained as colorless liquid (yield 72%).

¹H NMR (300 MHz, CDCl₃): δ1.42 (s, 9H), 1.70-1.84 (m, 2H), 1.76 (d, 3H,J=6.6 Hz), 1.92 (s, 3H), 2.64-2.80 (m, 1H), 3.22-3.35 (m, 3H), 4.58-4.64(m, 1H), 4.80-4.98 (br, 1H), 5.72-5.82 (m, 1H).

(Step 3)

In the same manner as in Example 1, Step 2, and by usingdihydroisoxazole 6Aa-03 as a substrate, N-Boc aminohydroxyketone 5Aa-003was obtained as colorless liquid (yield 66%).

¹H NMR (300 MHz, CDCl₃): δ1.42 (s, 9H), 1.50-1.70 (m, 2H), 1.76 (s, 3H),1.84 (d, 3H, J=6.6 Hz), 2.72-2.82 (m, 2H), 3.10-3.42 (m, 2H), 4.12-4.20(m, 1H), 4.96-5.10 (br, 1H), 6.70-6.82 (m, 1H).

(Steps 4 and 5)

In the same manner as in Example 1, Steps 4 and 5, and by using N-Bocaminohydroxyketone 5Aa-003 as a substrate, a mixture ofaminohydroxyketone 4Aa-003 and cyclic hemiaminal 3Aa-003 was obtained asyellow liquid (yield 82%).

¹H NMR (300 MHz, CDCl₃): δ1.48-1.72 (m, 2H), 1.76 (s, 3H), 1.84 (d, 3H,J=6.6 Hz), 2.72-2.82 (m, 2H), 2.96-3.22 (m, 2H), 4.02-4.12 (m, 1H),6.68-6.82 (m, 1H).

(steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4Aa-003 and cyclic hemiaminal 3Aa-003 as a substrate,cyclic imine 2Aa-003 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1Aa-003 was obtained as yellow liquid(yield 18%). MS: m/z 170 ([M+1], C₉H₁₅NO₂)

Example 8

(Step 1)

In the same manner as in Example 1, Step 1, and by using aldehyde 9a-04and hydroxylamine hydrochloride as substrates, oxime 8a-04 was obtainedas a colorless solid (yield 87%).

(Step 2)

In the same manner as in Example 1, Step 2, and by using oxime 8a-04 andN-Boc-aminobutene 7A-01 as substrates, dihydroisoxazole 6Aa-04 wasobtained as colorless liquid (yield 62%).

¹H NMR (300 MHz, CDCl₃): δ1.43 (s, 9H), 2.44-2.80 (m, 2H), 3.12-3.40 (m,4H), 3.94 (s, 2H), 4.62-4.76 (m, 1H), 5.10-5.18 (br, 1H), 6.80 (s, 1H),7.12-7.56 (m, 9H).

(Step 3)

In the same manner as in Example 1, Step 3, and by usingdihydroisoxazole 6Aa-04 as a substrate, N-Boc aminohydroxyketone 5Aa-004was obtained as colorless liquid (yield 65%).

¹H NMR (300 MHz, CDCl₃): δ1.43 (s, 9H), 2.46-2.84 (m, 2H), 3.14-3.42 (m,4H), 3.95 (s, 2H), 4.14-4.22 (m, 1H), 5.12-5.20 (br, 1H), 7.10-7.60 (m,10H).

(Steps 4 and 5)

In the same manner as in Example 1, Steps 4 and 5, and by using N-Bocaminohydroxyketone 5Aa-004 as a substrate, a mixture ofaminohydroxyketone 4Aa-004 and cyclic hemiaminal 3Aa-004 was obtained asyellow liquid (yield 80%).

¹H NMR (300 MHz, CDCl₃): δ2.46-2.84 (m, 2H), 3.12-3.42 (m, 4H), 3.94 (s,2H), 4.08-4.20 (m, 1H), 6.20 (s, 1H), 7.12-7.60 (m, 9H).

(steps 6 and 7)

In the same manner as in Example 1, Step 6, and by using a mixture ofaminohydroxyketone 4Aa-004 and cyclic hemiaminal 3Aa-004 as a substrate,cyclic imine 2Aa-004 was obtained. In the same manner as in Example 1,Step 7, bicyclic oxaziridine 1Aa-004 was obtained as yellow liquid(yield 15%). MS: m/z 342 ([M+1], C₂₀H₂₀ClNO₂)

INDUSTRIAL APPLICABILITY

According to the present invention, a production method of kakeromycinand a derivative thereof showing an antifungal activity and cytotoxicityand expected as a new antifungal agent or anticancer agent, by chemicalsynthesis is provided.

This application is based on patent application No. 2015-039363 filed inJapan, the contents of which are encompassed in full herein.

The invention claimed is:
 1. A compound represented by formula (4):

wherein R is (1) a group represented by formula (A):

wherein R¹ and R² are the same or different and each is a C₁₋₂₀ alkylgroup, a C₆₋₂₀ aryl group optionally substituted by a halogen atom, or aC₇₋₂₀ aralkyl group, (2) a C₁₋₂₀ alkyl group, (3) a C₆₋₂₀ aryl group, or(4) a C₇₋₂₀ aralkyl group, and n is 1, or a salt thereof.
 2. Thecompound of claim 1, wherein R is a group represented by formula (A):

wherein R¹ and R² are the same or different and each is a C₁₋₂₀ alkylgroup, a C₆₋₂₀ aryl group optionally substituted by a halogen atom, or aC₇₋₂₀ aralkyl group, or a salt thereof.
 3. The compound of claim 1,wherein R is a C₁₋₂₀ alkyl group, or a salt thereof.
 4. The compound ofclaim 1, wherein R is a C₆₋₂₀ aryl group, or salt thereof.
 5. Thecompound of claim 1, wherein R is a C₇₋₂₀ aralkyl group, or a saltthereof.
 6. A compound represented by formula (5):

wherein R is an optionally substituted heterocyclic group, R⁴ is anoptionally substituted hydrocarbon-oxy group, and n is 0, or a saltthereof.
 7. A compound represented by formula (5):

wherein R is an optionally substituted hydrocarbon group, R⁴ is anoptionally substituted hydrocarbon-oxy group, and n is 1, or a saltthereof.
 8. A compound represented by formula (5):

wherein R is an optionally substituted heterocyclic group, R⁴ is anoptionally substituted hydrocarbon group or an optionally substitutedhydrocarbon-oxy group, and n is 1, or a salt thereof.
 9. The compound ofclaim 8, wherein R⁴ is an optionally substituted hydrocarbon group, or asalt thereof.
 10. The compound of claim 8, wherein R⁴ is an optionallysubstituted hydrocarbon-oxy group, or a salt thereof.